High-purity steviol glycosides

ABSTRACT

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and X are described. The method includes expression of UDP-glucosyltransferases from  Stevia rebaudiana  Bertoni, which are capable converting certain steviol glycosides to rebaudiosides A, D and X. The highly purified rebaudiosides A, D and X, are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation application of PCT ApplicationNo. PCT/US2013/030439, filed Mar. 12, 2013, which claims the benefit ofU.S. Provisional Application No. 61/649,978, filed May 22, 2013. Thecontents of each of these applications are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention relates to a biocatalytic process for preparingcompositions comprising steviol glycosides, including highly purifiedsteviol glycoside compositions.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The text file entitled “PureCircle 22PCT ST25.txt,” created on Mar. 12,2013, having 5 KB (kilobytes) of data, and filed concurrently herewith,is hereby incorporated by reference in its entirety in this application.

BACKGROUND OF THE INVENTION

High intensity sweeteners possess a sweetness level that is many timesgreater than the sweetness level of sucrose. They are essentiallynon-caloric and are commonly used in diet and reduced-calorie products,including foods and beverages. High intensity sweeteners do not elicit aglycemic response, making them suitable for use in products targeted todiabetics and others interested in controlling for their intake ofcarbohydrates.

Steviol glycosides are a class of compounds found in the leaves ofStevia rebaudiana Bertoni, a perennial shrub of the Asteraceae(Compositae) family native to certain regions of South America. They arecharacterized structurally by a single base, steviol, differing by thepresence of carbohydrate residues at positions C13 and C19. Theyaccumulate in Stevia leaves, composing approximately 10%-20% of thetotal dry weight. On a dry weight basis, the four major glycosides foundin the leaves of Stevia typically include stevioside (9.1%),rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%).Other known steviol glycosides include rebaudioside B, C, D, E, F and X,steviolbioside and rubusoside.

Although methods are known for preparing steviol glycosides from Steviarebaudiana, many of these methods are unsuitable for use commercially.

Accordingly, there remains a need for simple, efficient, and economicalmethods for preparing compositions comprising steviol glycosides,including highly purified steviol glycoside compositions.

SUMMARY OF THE INVENTION

The present invention provides a biocatalytic process for preparing acomposition comprising a target steviol glycoside by contacting astarting composition comprising a steviol glycoside substrate withUDP-glucosyltransferase, thereby producing a composition comprising atarget steviol glycoside comprising one or more additional glucose unitsthan the steviol glycoside substrate.

The starting composition can be any composition comprising at least onesteviol glycoside substrate. In one embodiment, the steviol glycosidesubstrate is selected from the group consisting of steviolmonoside,steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B,rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudiosideA, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L,rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D,rebaudioside N, rebaudioside O, a synthetic steviol glycoside orcombinations thereof. The starting composition may be commerciallyavailable or prepared. The starting composition may comprise a purifiedsteviol glycoside substrate or a partially purified steviol glycosidesubstrate.

In one embodiment, the steviol glycoside substrate is rubusoside.

In another embodiment, the steviol glycoside substrate is stevioside.

In still another embodiment, the steviol glycoside substrate isrebaudioside A.

In yet another embodiment, the steviol glycoside substrate isrebaudioside D.

The target steviol glycoside can be any known steviol glycoside. In oneembodiment, the target steviol glycoside is steviolmonoside,steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B,rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudiosideA, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L,rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D,rebaudioside N, rebaudioside O or a synthetic steviol glycoside.

In one embodiment, the target steviol glycoside is stevioside.

In another embodiment, the target steviol glycoside is rebaudioside A.

In still another embodiment, the target steviol glycoside isrebaudioside D.

In yet another embodiment, the target steviol glycoside is rebaudiosideX

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capableof adding at least one glucose unit to the steviol glycoside substrateto provide the target steviol glycoside. In one embodiment,UDP-glucosyltransferase is produced in a host. The host may be, forexample, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp. Inanother embodiment, the UDP-glucosyltransferase is synthesized.

The UDP-glucosyltransferase can be provided in any suitable form,including free, immobilized or as a whole cell system. The degree ofpurity of the UDP-glucosyltransferase may vary, e.g., it may be providedas a crude, semi-purified or purified enzyme preparation(s).

In one embodiment, the UDP-glucosyltransferase is free. In anotherembodiment, the UDP-glucosyltransferase is immobilized, for example onan inorganic or organic support. In yet another embodiment, theUDP-glucosyltransferase is provided in the form of a whole cell system,for example as a living microbial cell, or in the form of a cell lysate.

In one embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torubusoside to form stevioside. In a particular embodiment, theUDP-glucosyltransferase is UGT91D2.

In one embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit tostevioside to form rebaudioside A. In a particular embodiment, theUDP-glucosyltransferase is UGT76G1.

In another embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torebaudioside A to form rebaudioside D. In a particular embodiment, theUDP-glucosyltransferase is UGT91D2.

In yet another embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torebaudioside D to form rebaudioside X. In a particular embodiment, theUDP-glucosyltransferase is UGT76G1.

Optionally, the method of the present invention further comprisesrecycling UDP to provide UDP-glucose. In one embodiment, the methodcomprises recycling UDP by providing a recycling catalyst and arecycling substrate, such that the biotransformation of the steviolglycoside substrate to the target steviol glycoside is carried out usingcatalytic amounts of UDP-glucosyltransferase and UDP-glucose (FIG. 3).

In one embodiment, the recycling catalyst is sucrose synthase.

In one embodiment, the recycling substrate is sucrose.

Optionally, the method of the present invention further comprisesseparating the target steviol glycoside from the starting composition.The target steviol glycoside can be separated by any suitable method,such as, for example, crystallization, separation by membranes,centrifugation, extraction, chromatographic separation or a combinationof such methods.

In one embodiment, separation produces a composition comprising greaterthan about 80% by weight of the target steviol glycoside on an anhydrousbasis, i.e., a highly purified steviol glycoside composition. In anotherembodiment, separation produces a composition comprising greater thanabout 90% by weight of the target steviol glycoside. In particularembodiments, the composition comprises greater than about 95% by weightof the target steviol glycoside.

The target steviol glycoside can be in any polymorphic or amorphousform, including hydrates, solvates, anhydrous or combinations thereof.

Purified target steviol glycosides can be used in consumable products asa sweetener. Suitable consumer products include, but are not limited to,food, beverages, pharmaceutical compositions, tobacco products,nutraceutical compositions, oral hygiene compositions, and cosmeticcompositions.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows the structure of reb X

FIG. 2 shows the biocatalytic production of reb X from stevioside.

FIG. 3 shows the biocatalytic production of reb A from stevioside usingthe enzyme UGT76G1 and concomitant recycling of UDP to UDP glucose viasucrose synthase.

FIG. 4 shows the IR spectrum of reb X.

FIG. 5. shows the HPLC chromatogram of the product of the biocatalyticproduction of reb X from reb D, as detailed in Example 14. The peak withretention time of 24.165 minutes corresponds to unreacted reb D. Thepeak with retention time of 31.325 minutes corresponds to reb X.

FIG. 6. shows the HPLC chromatogram of purified reb X produced bybiocatalysis from reb D.

FIG. 7 shows the HPLC chromatogram of a reb X standard.

FIG. 8 shows the HPLC chromatogram of co-injection of a reb X standardand reb X purified from biotransformation from reb D.

FIG. 9 shows an overlay of the ¹H NMR spectra of a reb X standard andreb X purified following biosynthesis from reb D.

FIG. 10 shows the HRMS spectrum of reb X purified following biocatalyticproduction from reb D.

FIG. 11 is a graph showing UGT76G1 catalyzed transformation ofstevioside to Reb A measured by CAD.

FIG. 12 is a graph showing the synthesis of Rebaudioside X fromRebaudioside D catalyzed by in-vitro produced UGT76G1 using CADdetection.

DETAILED DESCRIPTION

The present invention provides a biocatalytic process for thepreparation of a composition comprising a target steviol glycoside froma starting composition comprising a steviol glycoside substrate, whereinthe target steviol glycoside comprises one or more additional glucoseunits than the steviol glycoside substrate.

One object of the invention is to provide an efficient biocatalyticmethod for preparing steviol glycosides, particularly stevioside, reb A,reb D and reb X, from other steviol glycosides and/or mixtures thereof.

As used herein, “biocatalysis” or “biocatalytic” refers to the use ofnatural catalysts, such as protein enzymes, to perform chemicaltransformations on organic compounds. Biocatalysis is alternativelyknown as biotransformation or biosynthesis. Both isolated and whole-cellbiocatalysis methods are known in the art. Biocatalyst protein enzymescan be naturally occurring or recombinant proteins.

As used herein, the term “steviol glycoside(s)” refers to a glycoside ofsteviol, including, but not limited to, naturally occurring steviolglycosides, e.g. steviolmonoside, steviolbioside, rubusoside, dulcosideB, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudiosideC, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E,rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J,rebaudioside X, rebaudioside D, rebaudioside N, rebaudioside O,synthetic steviol glycosides, e.g. enzymatically glucosylated steviolglycosides and combinations thereof.

Chemical structures of steviol and its glycosides

Compound R₁ R₂ Steviol H H Steviolmonoside H β-Glc Steviol monoglucosylester β-Glc H Rubusoside β-Glc β-Glc Steviolbioside H β-Glc-β-Glc (2→1)Stevioside β-Glc β-Glc-β-Glc (2→1) Rebaudioside A β-Glc β-Glc-β-Glc(2→1)  | β-Glc (3→1) Rebaudioside D β-Glc-β-Glc (2→1) β-Glc-β-Glc (2→1) | β-Glc (3→1) Rebaudioside X β-Glc-β-Glc (2→1) β-Glc-β-Glc (2→1)  |  |β-Glc (3→1) β-Glc (3→1) (Glc = glucose)

Starting Composition

As used herein, “starting composition” refers to any composition(generally an aqueous solution) containing one or more steviolglycosides, where the one or more steviol glycosides serve as thesubstrate for the biotransformation.

In one embodiment, the starting composition comprises one or moresteviol glycosides selected from the group consisting ofsteviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A,rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudiosideF, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H,rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X,rebaudioside D, rebaudioside N, rebaudioside O or a synthetic steviolglycoside. In a particular embodiment, the starting compositioncomprises two or more steviol glycosides.

In one embodiment, the starting composition comprises the steviolglycoside substrate rubusoside.

In one embodiment, the starting composition comprises the steviolglycoside substrate stevioside.

In another embodiment, the starting composition comprises the steviolglycoside substrate rebaudioside A.

In yet another embodiment, the starting composition comprises thesteviol glycoside substrate rebaudioside D.

The starting composition may be synthetic or purified (partially orentirely), commercially available or prepared. One example of a startingcomposition useful in the method of the present invention is an extractobtained from purification of Stevia rebaudiana plant material (e.g.leaves). Another example of a starting composition is a commerciallyavailable stevia extract brought into solution with a solvent. Yetanother example of a starting composition is a commercially availablemixture of steviol glycosides brought into solution with a solvent.Other suitable starting compositions include by-products of processes toisolate and purify steviol glycosides.

In one embodiment, the starting composition comprises a purified steviolglycoside substrate. For example, the starting composition may comprisegreater than about 99% of a particular substrate steviol glycoside byweight on a dry basis.

In another embodiment, the starting composition comprises a partiallypurified substrate steviol glycoside composition. For example, thestarting composition contains greater than about 50%, about 60%, about70%, about 80% or about 90% of a particular substrate steviol glycosideby weight on a dry basis.

In one embodiment, the starting composition comprises purifiedrubusoside. In a particular embodiment, the starting compositioncontains >99% rubusoside by weight on a dry basis. In anotherembodiment, the starting composition comprises partially purifiedrubusoside. In a particular embodiment, the starting compositioncontains greater than about 50%, about 60%, about 70%, about 80% orabout 90% rubusoside by weight on a dry basis.

In one embodiment, the starting composition comprises purifiedstevioside. In a particular embodiment, the starting compositioncontains >99% stevioside by weight on a dry basis. In anotherembodiment, the starting composition comprises partially purifiedstevioside. In a particular embodiment, the starting compositioncontains greater than about 50%, about 60%, about 70%, about 80% orabout 90% stevioside by weight on a dry basis.

In another embodiment, the starting composition comprises purifiedrebaudioside A. In a particular embodiment, the starting compositioncontains greater than about 99% rebaudioside A by weight on a dry basis.In another embodiment, the starting composition comprises partiallypurified rebaudioside A. In a particular embodiment, the startingcomposition contains greater than about 50%, about 60%, about 70%, about80% or about 90% rebaudioside A by weight on a dry basis.

In yet another embodiment, the starting composition comprises purifiedrebaudioside D. In a particular embodiment, the starting compositioncontains greater than about 99% rebaudioside D by weight on a dry basis.In another embodiment, the starting composition comprises partiallypurified rebaudioside D. In a particular embodiment, the startingcomposition contains greater than about 50%, about 60%, about 70%, about80% or about 90% rebaudioside D by weight on a dry basis.

The steviol glycoside component(s) of the starting composition serve asa substrate(s) for the production of the target steviol glycoside(s), asdescribed herein. The target steviol glycoside target(s) differschemically from its corresponding steviol glycoside substrate(s) by theaddition of one or more glucose units.

Target Steviol Glycoside

The target steviol glycoside of the present method can be any steviolglycoside that can be prepared by the process disclosed herein. In oneembodiment, the target steviol glycoside is selected from the groupconsisting of steviolmonoside, steviolbioside, rubusoside, dulcoside B,dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C,rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E,rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J,rebaudioside X, rebaudioside D, rebaudioside N or rebaudioside O.

In one embodiment, the target steviol glycoside is stevioside. Inanother embodiment, the target steviol glycoside is reb A. In yetanother embodiment, the target steviol glycoside is reb D. In stillanother embodiment, the target steviol glycoside is reb X.

The target steviol glycoside can be in any polymorphic or amorphousform, including hydrates, solvates, anhydrous or combinations thereof.

In one embodiment, the present invention is a biocatalytic process forthe production of stevioside from rubusoside, where the startingcomposition comprises the steviol glycoside substrate rubusoside. In aparticular embodiment, the present invention is a biocatalytic processfor the production of stevioside from rubusoside, where the startingcomposition comprises partially purified rubusoside. In anotherparticular embodiment, the present invention is a biocatalytic processfor the production of stevioside from rubusoside, where the startingcomposition comprises purified rubusoside.

In one embodiment, the present invention is a biocatalytic process forthe production of reb A from stevioside, where the starting compositioncomprises the steviol glycoside substrate stevioside. In a particularembodiment, the present invention is a biocatalytic process for theproduction of reb A from stevioside, where the starting compositioncomprises partially purified stevioside. In another particularembodiment, the present invention is a biocatalytic process for theproduction of reb A from stevioside, where the starting compositioncomprises purified stevioside.

In another embodiment, the present invention is a biocatalytic processfor the production of reb D from reb A, where the starting compositioncomprises the steviol glycoside substrate reb A. In a particularembodiment, the present invention is a biocatalytic process for theproduction of reb D from reb A, where the starting composition comprisespartially purified reb A. In another particular embodiment, the presentinvention is a biocatalytic process for the production of reb D from rebA, where the starting composition comprises purified reb A.

In still another embodiment, the present invention is a biocatalyticprocess for the production of reb X from reb D, where the startingcomposition comprises the steviol glycoside substrate reb D. In aparticular embodiment, the present invention is a biocatalytic processfor the production of reb X from reb D, where the starting compositioncomprises partially purified reb D. In another particular embodiment,the present invention is a biocatalytic process for the production ofreb X from reb D, where the starting composition comprises purified rebD.

In a particular embodiment, the target steviol glycoside is present in amixture. For example, in one embodiment, the target steviol glycoside isreb X present in a mixture. In one embodiment, the purity of the targetsteviol glycoside is increased relative to the purity of the targetsteviol glycoside present in the starting composition. For example, thepurity of reb X present in the starting composition is increased as aresult of carrying out the method of the present invention.

Optionally, the method of the present invention further comprisesseparating the target steviol glycoside from the starting composition.The target steviol glycoside can be separated by any suitable method,such as, for example, crystallization, separation by membranes,centrifugation, extraction, chromatographic separation or a combinationof such methods.

In particular embodiments, the process described herein results in ahighly purified target steviol glycoside composition. The term “highlypurified”, as used herein, refers to a composition having greater thanabout 80% by weight of the target steviol glycoside on an anhydrousbasis. In one embodiment, the highly purified target steviol glycosidecomposition contains greater than about 90% by weight of the targetsteviol glycoside on an anhydrous basis, such as, for example, 91%greater than about 92%, greater than about 93%, greater than about 94%,greater than about 95%, greater than about 95%, greater than about 97%,greater than about 98% or greater than about 99% target steviolglycoside content on a dry basis.

In a more particular embodiment, when the target steviol glycoside isreb X, the process described herein provides a composition havinggreater than about 90% reb X content by weight on a dry basis. Inanother particular embodiment, when the target steviol glycoside is rebX, the process described herein provides a composition comprisinggreater than about 95% reb X content by weight on a dry basis.

In another particular embodiment, when the target steviol glycoside isreb D, the process described herein provides a composition greater thanabout 90% reb D content by weight on a dry basis. In another particularembodiment, when the target steviol glycoside is reb D, the processdescribed herein provides a composition comprising greater than about95% reb D content by weight on a dry basis.

In still another particular embodiment, when the target steviolglycoside is reb A, the process described herein provides a compositioncomprising greater than about 90% reb A content by weight on a drybasis. In another particular embodiment, when the target steviolglycoside is reb A, the process described herein provides a compositioncomprising greater than about 95% reb A content by weight on a drybasis.

In yet another particular embodiment, when the target steviol glycosideis stevioside, the process described herein provides a compositioncomprising greater than about 90% stevioside content by weight on a drybasis. In another particular embodiment, when the target steviolglycoside is stevioside, the process described herein provides acomposition comprising greater than about 95% stevioside content byweight on a dry basis.

In one embodiment, the biocatalytic method of the present invention iscarried out more than one time, such that the target steviol glycosideproduced by a first biocatalytic process serves as the steviol glycosidesubstrate (which could also be considered an intermediate target steviolglycoside) for a second biocatalytic process in which the target steviolglycoside is produced.

In a particular embodiment, the present invention provides abiocatalytic process for preparing a composition comprising a targetsteviol glycoside by contacting a starting composition comprising asteviol glycoside substrate with a UDP-glucosyltransferase, therebyproducing a composition comprising an intermediate target steviolglycoside comprising one or more additional glucose units than thesteviol glycoside substrate; contacting the composition comprising theintermediate target steviol glycoside with UDP-glucosyltransferase,thereby producing a target steviol glycoside comprising one or moreadditional glucose units than the intermediate target steviol glycoside.Depending on the number of times the method is carried out, there may beone or more intermediate target steviol glycosides (e.g., a firstintermediate target steviol glycoside, a second intermediate targetsteviol glycoside, a third intermediate target steviol glycoside)involved in the production of the target steviol glycoside.

UDP-Glucotransferase

The present method is biocatalytic, i.e., utilizes a biologicalcatalyst. In one embodiment, the biocatalyst is protein enzyme. In aparticular embodiment, the biocatalyst is a UDP-glucosyltransferase. TheUDP-glucosyltransferase can be any UDP-glucosyltransferase capable ofadding at least one glucose unit to the steviol glycoside substrate toprovide the target steviol glycoside.

In one embodiment, the UDP-glucosyltransferase is produced in a host,such as a microorganism. For example, a DNA sequence encodingUDP-glucosyltransferase is cloned into an expression vector andtransferred into a production host such as a microbe, e.g., a bacteria.Non-limiting examples of suitable hosts include E. coli, Saccharomycessp., Aspergillus sp., Pichia sp. The overexpressed protein can beisolated from the cell extract based on its physical and chemicalproperties, using techniques known in the art. Representativenon-limiting techniques for isolating UDP-glucosyltransferase from ahost include centrifugation, electrophoresis, liquid chromatography, ionexchange chromatography, gel filtration chromatography or affinitychromatography.

UDP-glucosyltransferase can be provided as a crude, semi-purified andpurified enzyme preparation(s).

In one embodiment, the UDP-glucosyltransferase is free. In anotherembodiment, the UDP-glucosyltransferase is immobilized. For example,UDP-glucosyltransferase may be immobilized to a solid support made frominorganic or organic materials. Non-limiting examples of solid supportssuitable to immobilize UDP-glucosyltransferase include derivatizedcellulose or glass, ceramics, metal oxides or membranes.UDP-glucosyltransferase may be immobilized to the solid support, forexample, by covalent attachment, adsorption, cross-linking, entrapmentor encapsulation.

The reaction medium for conversion is generally aqueous, e.g., purifiedwater, buffer or a combination thereof. In a particular embodiment, thereaction medium is a buffer. Suitable buffers include, but are notlimited to, PIPES buffer, acetate buffer and phosphate buffer. In aparticular embodiment, the reaction medium is a phosphate buffer. Thereaction medium can also be, alternatively, an organic solvent.

In one embodiment, the UDP-glucosyltransferase is provided in the formof a whole cell system, such as a living microbial cell. The whole cellsystem may optionally be immobilized, as well, utilizing the techniquesidentified above with respect to immobilization of the enzyme.

In one embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torubusoside, thereby producing stevioside. The UDP-glucosyltransferasemay be, for example, UGT91 D2.

In another embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit tostevioside, thereby producing rebaudioside A. TheUDP-glucosyltransferase may be, for example, UGT76G1.

In still another embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torebaudioside A, thereby producing rebaudioside D. TheUDP-glucosyltransferase may be, for example, UGT91D2.

In yet another embodiment, the UDP-glucosyltransferase is anyUDP-glucosyltransferase capable of adding at least one glucose unit torebaudioside D to form rebaudioside X. The UDP-glucosyltransferase maybe, for example, UGT76G1.

Optionally, the method of the present invention further comprisesrecycling UDP to provide UDP-glucose. In one embodiment, the methodcomprises recycling UDP by providing a recycling catalyst, i.e., abiocatalyst capable of UDP-glucose overproduction, and a recyclingsubstrate, such that the conversion of the substrate steviol glycosideto the target steviol glycoside is carried out using catalytic amountsof UDP-glucosyltransferase and UDP-glucose (FIG. 3).

In one embodiment, the UDP-glucose recycling catalyst is sucrosesynthase.

In one embodiment, the recycling substrate is sucrose.

The Conversion of Rubusoside to Stevioside

In one embodiment, a starting composition comprising rubusoside iscontacted with a UDP-glucosyltransferase capable of catalyzing thereaction of UDP-glucose and stevioside to produce stevioside. In oneembodiment, the starting composition comprises partially purifiedrubusoside. In another embodiment, the starting composition comprisespurified rubusoside. In a particular embodiment, the startingcomposition comprises >99% rubusoside. In a particular embodiment, thestarting composition comprises greater than about 50%, about 60%, about70% about 80% or about 90% rubusoside.

In a particular embodiment, the UDP-glucosyltransferase is UGT91D2,which has been described by Joseph et al. (Genbank accession no.ACE87855). It has to be noted that similar sequence was described laterin a patent application PCT/US2011/038967 and named UGT91D2e. UGT91D2eshares >95% identity with UGT91D11 (Genbank accession no. AAR06918)and >99% identity with UGT of Joseph et al. (Genbank accession no.ACE87855).

In some embodiments, the UDP-glucosyltransferase, such as UGT91D2, isprepared by expression in a host microorganism. Suitable hostmicroorganisms include, but are not limited to, E. coli, Saccharomycessp., Aspergillus sp., Pichia sp. In a particular embodiment, UGT91D2 isexpressed in E. coli.

The UDP-glucosyltransferase, such as UGT91D2, can be provided free or inan immobilized form. The enzyme preparation may be crude, semi-purifiedand purified. In one embodiment, the UDP-glucosyltransferase is providedas a whole-cell system, e.g., a living microbial cell, or wholemicrobial cells, cell lysate and/or any other form of known in the art.

The reaction medium for conversion is generally aqueous, and can bepurified water, buffer or a combination thereof. In a particularembodiment, the reaction medium is a buffer. Suitable buffers include,but are not limited to, PIPES buffer, acetate buffer and phosphatebuffer. In one embodiment, the reaction medium is phosphate buffer.

In one embodiment, conversion of rubusoside to stevioside furthercomprises the addition of compounds other than UDP-glucose, rubusosideand the UDP-glucosyltranferase. For example, in some embodiments, thereaction medium includes MgCl₂ and/or MnCl₂.

The reaction can be carried out at temperature between about 0° C. andabout 60° C., such as, for example, about 10° C., about 20° C., about30° C., about 40° C., about 50° C. or about 60° C. In a particularembodiment, the reaction is carried out at about 30° C.

The reaction can proceed for a duration of time between 1 hour and 1week, such as, for example, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, about 120 hours, about 3 days,about 4 days, about 5 days, about 6 days or about 7 days. In aparticular embodiment, the reaction is carried out for about 120 hours.

Optionally, the UDP-glucose, which is used as glucose donor, can berecycled by the use of the enzyme sucrose synthase (FIG. 3). Rubusosideis transformed into stevioside with UDP-glucose which is recycled by thereaction between sucrose and UDP. As a consequence, rubusoside andsucrose are used in stoichiometric amounts whereas UDP is present incatalytic amounts.

The reaction can be monitored by suitable method including, but notlimited to, HPLC, LCMS, TLC, IR or NMR.

In one embodiment, the conversion of rubusoside to stevioside is atleast about 2% complete, as determined by any of the methods mentionedabove. In a particular embodiment, the conversion of rubusoside tostevioside is at least about 10% complete, at least about 20% complete,at least about 30% complete, at least about 40% complete, at least about50% complete, at least about 60% complete, at least about 70% complete,at least about 80% complete or at least about 90% complete. In aparticular embodiment, the conversion of rubusoside to stevioside is atleast about 95% complete.

The Conversion of Stevioside to Reb A

In one embodiment, a starting composition comprising stevioside iscontacted with a UDP-glucosyltransferase capable of catalyzing thereaction of UDP-glucose and stevioside to produce reb A. Chemically, aglucose unit is added to the disaccharide at the C13 position ofstevioside to provide reb A. In one embodiment, the starting compositioncomprises partially purified stevioside. In another embodiment, thestarting composition comprises purified stevioside. In a particularembodiment, the starting composition comprises >99% stevioside. In aparticular embodiment, the starting composition comprises greater thanabout 50%, about 60%, about 70% about 80% or about 90% stevioside.

In a particular embodiment, the UDP-glucosyltransferase is UGT76G1.UGT76G1 has been described by Richman et al. (Richman, A., Swanson, A.,Humphrey, T., Chapman, R., McGarvey, B., Pocs, R., Brandle, J.Functional genomics uncovers three glucosyltransferases involved in thesynthesis of the major sweet glucosides of Stevia rebaudiana. The PlantJournal, 2005, 41, 56-67) and is accessible in Genbank (ACT33422.1) andUniprot (C7EA09). The enzyme was overexpressed in E. coli and was shownto transform stevioside to reb A.

In some embodiments, the UDP-glucosyltransferase, such as UGT76G1, canbe prepared by expression in a host microorganism. Suitable hostmicroorganisms include, but are not limited to, E. coli, Saccharomycessp., Aspergillus sp., Pichia sp. In a particular embodiment, UGT76G1 isexpressed in E. coli.

The UDP-glucosyltransferase, such as UGT76G1, can be free orimmobilized. It can be in the form of crude, semi-purified and purifiedenzyme preparation(s). The UDP-glucosyltransferase can also be providedas a whole cell system, e.g., a living microbial cell, a whole microbialcell or cell lysate and/or any other form of known in the art.

The reaction medium for conversion is generally aqueous, and can bepurified water, buffer or a combination thereof. In a particularembodiment, the reaction medium is a buffer. Suitable buffers include,but are not limited to, PIPES buffer, acetate buffer and phosphatebuffer. In one embodiment, the reaction medium is phosphate buffer.

In one embodiment, conversion of stevioside to reb A further comprisingthe addition of compounds other than UDP-glucose, stevioside and theUDP-glucosyltranferase. For example, in some embodiments, the reactionmedium includes MgCl₂ and/or MnCl₂.

The reaction can be carried out at temperature between about 0° C. andabout 60° C., such as, for example, about 10° C., about 20° C., about30° C., about 40° C., about 50° C. or about 60° C. In a particularembodiment, the reaction is carried out at about 30° C.

The reaction can proceed for a duration of time between 1 hour and 1week, such as, for example, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, about 120 hours, about 3 days,about 4 days, about 5 days, about 6 days or about 7 days. In aparticular embodiment, the reaction is carried out for about 120 hours.

Optionally, the UDP-glucose, which is used as glucose donor, can berecycled by the use of the enzyme sucrose synthase (FIG. 3). Steviosideis transformed into reb A with UDP-glucose which is recycled by thereaction between sucrose and UDP. As a consequence, stevioside andsucrose are used in stoichiometric amounts whereas UDP is present incatalytic amounts.

The reaction can be monitored by suitable method including, but notlimited to, HPLC, LCMS, TLC, IR or NMR.

In one embodiment, the biocatalytic conversion or biotransformation ofstevioside to reb A is at least about 50% complete, as determined by anyof the methods mentioned above. In a particular embodiment, theconversion of stevioside to reb A is at least about 60% complete, atleast about 70% complete, at least about 80% complete or at least about90% complete. In a particular embodiment, the conversion of steviosideto reb A is at least about 95% complete.

The Conversion of Reb A to Reb D

In one embodiment, a starting composition comprising reb A is contactedwith a UDP-glucosyltransferase capable of catalyzing the reaction ofUDP-glucose and reb A to produce reb D. Chemically, a glucose unit isadded to the monosaccharide at the C19 position of reb A to provide rebD. In one embodiment, the starting composition comprises partiallypurified reb A. In another embodiment, the starting compositioncomprises purified reb A. In a particular embodiment, the startingcomposition comprises >99% reb A. In a particular embodiment, thestarting composition comprises greater than about 50%, about 60%, about70% about 80% or about 90% reb A.

In a particular embodiment, the UDP-glucosyltransferase is UGT91D2,which has been described by Joseph et al. (Genbank accession no.ACE87855). It has to be noted that similar sequence was described laterin a patent application PCT/US2011/038967 and named UGT91D2e. UGT91D2eshares >95% identity with UGT91D11 (Genbank accession no. AAR06918)and >99% identity with UGT of Joseph et al. (Genbank accession no.ACE87855).

In some embodiments, the UDP-glucosyltransferase, such as UGT91D2, isprepared by expression in a host microorganism. Suitable hostmicroorganisms include, but are not limited to, E. coli, Saccharomycessp., Aspergillus sp., Pichia sp. In a particular embodiment, UGT91D2 isexpressed in E. coli.

The UDP-glucosyltransferase, such as UGT91D2, can be provided free or inan immobilized form. The enzyme preparation may be crude, semi-purifiedand purified. In one embodiment, the UDP-glucosyltransferase is providedas a whole-cell system, e.g., a living microbial cell, or wholemicrobial cells, cell lysate and/or any other form of known in the art.

The reaction medium for conversion is generally aqueous, and can bepurified water, buffer or a combination thereof. In a particularembodiment, the reaction medium is a buffer. Suitable buffers include,but are not limited to, PIPES buffer, acetate buffer and phosphatebuffer. In one embodiment, the reaction medium is phosphate buffer.

In one embodiment, conversion of reb A to reb D further comprising theaddition of compounds other than UDP-glucose, reb A and theUDP-glucosyltranferase. For example, in some embodiments, the reactionmedium includes MgCl₂ and/or MnCl₂.

The reaction can be carried out at temperature between about 0° C. andabout 60° C., such as, for example, about 10° C., about 20° C., about30° C., about 40° C., about 50° C. or about 60° C. In a particularembodiment, the reaction is carried out at about 30° C.

The reaction can proceed for a duration of time between 1 hour and 1week, such as, for example, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, about 120 hours, about 3 days,about 4 days, about 5 days, about 6 days or about 7 days. In aparticular embodiment, the reaction is carried out for about 120 hours.

Optionally, the UDP-glucose, which is used as glucose donor, can berecycled by the use of the enzyme sucrose synthase (FIG. 3). Reb A istransformed into reb D with UDP-glucose which is recycled by thereaction between sucrose and UDP. As a consequence, reb A and sucroseare used in stoichiometric amounts whereas UDP is present in catalyticamounts.

The reaction can be monitored by suitable method including, but notlimited to, HPLC, LCMS, TLC, IR or NMR.

In one embodiment, the conversion of reb A to reb D is at least about 2%complete, as determined by any of the methods mentioned above. In aparticular embodiment, the conversion of reb A to reb D is at leastabout 10% complete, at least about 20% complete, at least about 30%complete, at least about 40% complete, at least about 50% complete, atleast about 60% complete, at least about 70% complete, at least about80% complete or at least about 90% complete. In a particular embodiment,the conversion of reb A to reb D is at least about 95% complete.

The Conversion of Reb D to Reb X

In one embodiment, the starting composition comprises reb D, which iscontacted with a UDP-glucosyltransferase capable of catalyzing thereaction of UDP-glucose and reb D to produce reb X. Chemically, aglucose unit is added to the disaccharide at the C19 position of reb Dto provide reb X. In one embodiment, the starting composition comprisespartially purified reb D. In another embodiment, the startingcomposition comprises purified reb D. In a particular embodiment, thestarting composition comprises >99% reb D. In a particular embodiment,the starting composition comprises greater than about 50%, about 60%,about 70% about 80% or about 90% reb D. In a particular embodiment, theUDP-glucosyltransferase is UGT76G1.

In some embodiments, the UDP-glucosyltransferase, such as UGT91D2, canbe prepared by expression in a host microorganism. Suitable hostmicroorganisms include, but are not limited to, E. coli, Saccharomycessp., Aspergillus sp., Pichia sp. In a particular embodiment, UGT91D2 isexpressed in E. coli.

The UDP-glucosyltransferase, such as UGT91D2, can be provided as free orimmobilized. The enzyme preparation can be crude, semi-purified andpurified. In one embodiment, the UDP-glucosyltransferase is provided asa whole cell preparation, e.g., living microbial cells, or in the formof whole microbial cells, cell lysate and/or any other form of known inthe art.

The reaction medium for conversion is generally aqueous, and can bepurified water, buffer or a combination thereof. In a particularembodiment, the reaction medium is a buffer. Suitable buffers include,but are not limited to, PIPES buffer, acetate buffer and phosphatebuffer. In one embodiment, the reaction medium is phosphate buffer.

In one embodiment, conversion of reb D to reb X employs compounds inaddition to UDP-glucose, reb D and the UDP-glucosyltranferase. Forexample, in some embodiments, the reaction medium includes MgCl₂ and/orMnCl₂.

The reaction can be carried out at temperature between about 0° C. andabout 60° C., such as, for example, about 10° C., about 20° C., about30° C., about 40° C., about 50° C. or about 60° C. In a particularembodiment, the reaction is carried out at about 30° C.

The reaction can proceed for a duration of time between 1 hour and 1week, such as, for example, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, about 120 hours, about 3 days,about 4 days, about 5 days, about 6 days or about 7 days. In aparticular embodiment, the reaction is carried out for about 120 hours.

Optionally, the UDP-glucose, which is used as glucose donor, can berecycled by the use of the enzyme Sucrose Synthase (FIG. 3). Reb D istransformed into reb X with UDP-glucose which is recycled by thereaction between sucrose and UDP. As a consequence, reb D and sucroseare used in stoichiometric amounts whereas UDP is present in catalyticamounts.

The reaction can be monitored by suitable method including, but notlimited to, HPLC, LCMS, TLC, IR or NMR.

In one embodiment, the conversion of reb D to reb X is at least about50% complete, as determined by any of the methods mentioned above. In aparticular embodiment, the conversion of reb D to reb X is at leastabout 60% complete, at least about 70% complete, at least about 80%complete or at least about 90% complete. In a particular embodiment, theconversion of reb D to reb X is at least about 95% complete.

The target steviol glycoside is optionally purified from the resultingcomposition. Purification of the target steviol glycoside from thereaction medium can be achieved by any suitable method to provide ahighly purified target steviol glycoside composition. Suitable methodsinclude crystallization, separation by membranes, centrifugation,extraction (liquid or solid phase), chromatographic separation, HPLC(preparative or analytical) or a combination of such methods.

In one embodiment, the particular biocatalytic conversion can bequenched to stop the reaction. The resultant mixture is thencentrifuged. The supernatant generally contains the target steviolglycosides, and can then be further purified, if desired. For example,analytical or preparative HPLC can be used to separate remaining targetor starting steviol glycoside(s) or reaction by-products from the targetsteviol glycoside. In one embodiment, separation is achieved withanalytical HPLC. In another embodiment, separation is achieved withpreparative HPLC. One of skill in the art will recognize that theparticular HPLC method used can vary based on the particular system,solvent, and column. A suitable system for separating reb X from reb Dis provided in the Example 20.

It is envisaged that the method provided herein can be repeated, whereinthe composition resulting from the initial process, i.e., thecomposition comprising the target steviol glycoside, can then be used asthe starting composition when the method is carried out a second time-or optionally, the target steviol glycoside can be purified from thecomposition comprising the target steviol glycoside to provide a highlypurified target steviol glycoside or steviol glycoside composition.According to this embodiment, the target steviol glycoside produced whenthe method is carried out the first time can be considered a firsttarget steviol glycoside or an intermediate target steviol glycoside,useful as a substrate for the production of a second target steviolglycoside, a second intermediate target steviol glycoside or an ultimatetarget steviol glycoside. The process can be repeated as many times asrequired to arrive at the ultimate target steviol glycoside. In oneembodiment, the method is repeated once. In another embodiment, themethod is repeated twice. In yet another embodiment, the method isrepeated three times. In still other embodiments, the method is repeatedfour, five, six, seven, eight or nine times. On of skill in the art willrecognize that the particular UDP-glucosyltransferase used in eachreaction can either be the same or different, depending on theparticular site on the steviol glycoside substrate where glucose is tobe added.

Accordingly, in one embodiment, the method is repeated once, wherein thestarting composition of the first method comprises reb A and the targetsteviol glycoside is reb D, and wherein starting composition of thesecond method comprises reb D and the target steviol glycoside is reb X.

In another embodiment, the method is repeated twice, wherein thestarting composition of the first method comprises stevioside and thetarget steviol glycoside is reb A; the starting composition of thesecond method comprises reb A and the target steviol glycoside is reb D;and the starting composition of the third method comprises reb D and thetarget steviol glycoside is reb X.

In still another embodiment, the method is repeated three times, wherethe starting composition of the first method comprises rubusoside andthe target steviol glycoside is stevioside; the starting composition ofthe second method comprises stevioside and the target steviol glycosideis reb A; the starting composition of the third method comprises reb Aand the target steviol glycoside is reb D; and the starting compositionof the fourth method comprises reb D and the target steviol glycoside isreb X.

In one embodiment, a method for producing a highly purified targetsteviol glycoside composition comprises:

-   -   a. contacting a first starting composition comprising a steviol        glycoside substrate with a first UDP-glucosyltransferase to        produce a composition comprising a first target steviol        glycoside;    -   b. optionally separating the first target steviol glycoside from        the medium to provide a highly purified first target steviol        glycoside composition;    -   c. contacting the composition comprising a first target steviol        glycoside or the highly purified first target steviol glycoside        composition with a second UDP-glucosyltransferase to produce a        composition comprising a second target steviol glycoside;    -   d. optionally separating the second target steviol glycoside        from the medium to provide a highly purified second target        steviol glycoside composition;    -   e. contacting the composition comprising the second target        steviol glycoside or the highly purified second target steviol        glycoside composition with a third UDP-glucosyltransferase to        produce a composition comprising a third target steviol        glycoside; and    -   f. optionally separating the third target steviol glycoside from        the medium to provide a highly purified third target steviol        glycoside composition.

In one embodiment, the first starting composition comprises stevioside,the first target steviol glycoside is reb A, and the firstUDP-glucosyltransferase is UGT76G1.

In a further embodiment, the second UDP-glucosyltransferase is UGT91D2and the second target steviol glycoside is reb D.

In a still further embodiment, the third UDP-glucosyltransferase isUGT91D2 and the third target steviol glycoside is reb X

In one embodiment, one of more of the steps of contacting thecomposition comprising the steviol glycoside substrate withUDP-glucosyltransferase further includes providing a biocatalyst capableof UDP-overproduction and recycling and a substrate for said recycling.

In a more particular embodiment, a method for producing a highlypurified target steviol glycoside composition comprises:

-   -   a. contacting a first starting composition comprising a steviol        glycoside substrate with a first UDP-glucosyltransferase to        produce a composition comprising a first target steviol        glycoside;    -   b. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   c. optionally separating the first target steviol glycoside from        the medium to provide a highly purified first target steviol        glycoside composition;    -   d. contacting the composition comprising a first target steviol        glycoside or the highly purified first target steviol glycoside        composition with a second UDP-glucosyltransferase to produce a        composition comprising a second target steviol glycoside;    -   e. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   f. optionally separating the second target steviol glycoside        from the medium to provide a highly purified second target        steviol glycoside composition;    -   g. contacting the composition comprising the second target        steviol glycoside or the highly purified second target steviol        glycoside composition with a third UDP-glucosyltransferase to        produce a composition comprising a third target steviol        glycoside; and    -   h. optionally separating the third target steviol glycoside from        the medium to provide a highly purified third target steviol        glycoside composition.

In one embodiment, the first starting composition comprises stevioside,the first target steviol glycoside is reb A, and the firstUDP-glucosyltransferase is UGT76G1.

In a further embodiment, the second UDP-glucosyltransferase is UGT91D2and the second target steviol glycoside is reb D.

In a still further embodiment, the third UDP-glucosyltransferase isUGT91D2 and the third target steviol glycoside is reb X

In another particular embodiment, a method for producing a highlypurified target steviol glycoside composition comprises:

-   -   a. contacting a first starting composition comprising a steviol        glycoside substrate with a first UDP-glucosyltransferase to        produce a composition comprising a first target steviol        glycoside;    -   b. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   c. optionally separating the first target steviol glycoside from        the medium to provide a highly purified first target steviol        glycoside composition;    -   d. contacting the composition comprising a first target steviol        glycoside or the highly purified first target steviol glycoside        composition with a second UDP-glucosyltransferase to produce a        composition comprising a second target steviol glycoside;    -   e. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   f. optionally separating the second target steviol glycoside        from the medium to provide a highly purified second target        steviol glycoside composition;    -   g. contacting the composition comprising the second target        steviol glycoside or the highly purified second target steviol        glycoside composition with a third UDP-glucosyltransferase to        produce a composition comprising a third target steviol        glycoside; and    -   h. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   i. optionally separating the third target steviol glycoside from        the medium to provide a highly purified third target steviol        glycoside composition;    -   j. contacting the composition comprising the third target        steviol glycoside or the highly purified third target steviol        glycoside composition with a fourth UDP-glucosyltransferase to        produce a composition comprising a fourth target steviol        glycoside; and    -   k. optionally providing a biocatalyst capable of        UDP-overproduction and recycling and a substrate for said        recycling;    -   l. optionally separating the fourth target steviol glycoside        from the medium to provide a highly purified fourth target        steviol glycoside composition.

In one embodiment, the first starting composition comprises rubusoside,the first target steviol glycoside is stevioside, and the firstUDP-glucosyltransferase is UGT91D2.

In a further embodiment, the second UDP-glucosyltransferase is UGT76G1and the second target steviol glycoside is reb A.

In a further embodiment, the third UDP-glucosyltransferase is UGT91D2and the third target steviol glycoside is reb D.

In a still further embodiment, the fourth UDP-glucosyltransferase isUGT91D2 and the fourth target steviol glycoside is reb X.

Purified steviol glycosides, prepared in accordance with the presentinvention, may be used in a variety of products including, but notlimited to, foods, beverages, pharmaceutical compositions, tobaccoproducts, nutraceutical compositions, oral hygiene compositions, andcosmetic compositions.

The high purity reb X obtained in this invention, having a molecularweight of 1291.29, a molecular formula of C₅₆H₉₀O₃₃, and the structurepresented in FIG. 1, is in the form of a white and odorless powder. Thecompound is about 200 times sweeter than sugar when compared to a 10%sucrose solution. The infrared absorption spectrum is shown in FIG. 4.

Other properties of the pure reb X compound include a melting point of249-250° C., and a specific rotation of [α]_(D) ²⁵ −19.0° in 50% ethanol(C=1.0). The solubility of reb X in water is around 0.3%, and increaseswith an increase in temperature.

Reb X is soluble in diluted solutions of methanol, ethanol, n-propanol,and isopropanol. However, it is insoluble in acetone, benzene,chloroform, and ether.

Reb X obtained in accordance with the present invention is heat andpH-stable.

Highly purified target glycoside(s) particularly, reb D and/or reb Xobtained according to this invention can be used “as-is” or incombination with other sweeteners, flavors and food ingredients.

Non-limiting examples of flavors include lime, lemon, orange, fruit,banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon,sugar, cotton candy and vanilla flavors.

Non-limiting examples of other food ingredients include flavors,acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilisers, thickeners and gelling agents.

Highly purified target glycoside(s) particularly, reb D and/or reb Xobtained according to this invention can be prepared in variouspolymorphic forms, including but not limited to hydrates, solvates,anhydrous, amorphous forms and/or mixtures thereof.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X obtained according to this invention may be incorporated as a highintensity natural sweetener in foodstuffs, beverages, pharmaceuticalcompositions, cosmetics, chewing gums, table top products, cereals,dairy products, toothpastes and other oral cavity compositions, etc.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X as a sweetening compound may be employed as the sole sweetener, orit may be used together with other naturally occurring high intensitysweeteners such as stevioside, reb A, reb B, reb C, reb D, reb E, reb F,steviolbioside, dulcoside A, rubusoside, mogrosides, brazzein,neohesperidin dihydrochalcone, glycyrrhizic acid and its salts,thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside,phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid,abrusosides, periandrin, carnosiflosides, cyclocarioside,pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin,glycyphyllin, phlorizin, trilobatin, dihydroflavonol,dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatinand its salts, selligueain A, hematoxylin, monellin, osladin,pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin,curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener,mogroside V, siamenoside and others.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X may also be used in combination with synthetic high intensitysweeteners such as sucralose, potassium acesulfame, aspartame, alitame,saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin,suosan,N-[—N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N-[—N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester, salts thereof, and the like.

Moreover, highly purified target steviol glycoside(s), particularly, rebD and/or reb X can be used in combination with natural sweetenersuppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, andothers. Reb D and/or reb X may also be combined with various umami tasteenhancers. Reb D and/or reb X can be mixed with umami tasting and sweetaminoacids such as glutamate, aspartic acid, glycine, alanine,threonine, proline, serine, glutamate, and tryptophan.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X may also be combined with polyols or sugar alcohols. The term“polyol” refers to a molecule that contains more than one hydroxylgroup. A polyol may be a diol, triol, or a tetraol which contain 2, 3,and 4 hydroxyl groups, respectively. A polyol also may contain more thanfour hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like,which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, apolyol also may be a sugar alcohol, polyhydric alcohol, or polyalcoholwhich is a reduced form of carbohydrate, wherein the carbonyl group(aldehyde or ketone, reducing sugar) has been reduced to a primary orsecondary hydroxyl group. Examples of polyols include, but are notlimited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol,inositol, isomalt, propylene glycol, glycerol, threitol, galactitol,hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reducedxylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltosesyrup, reduced glucose syrup, hydrogenated starch hydrolyzates,polyglycitols and sugar alcohols or any other carbohydrates capable ofbeing reduced which do not adversely affect the taste of the sweetenercomposition.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X may be combined with reduced calorie sweeteners such asD-tagatose, L-sugars, L-sorbose, L-arabinose, and others.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X may also be combined with various carbohydrates. The term“carbohydrate” generally refers to aldehyde or ketone compoundssubstituted with multiple hydroxyl groups, of the general formula(CH₂O)_(n), wherein n is 3-30, as well as their oligomers and polymers.The carbohydrates of the present invention can, in addition, besubstituted or deoxygenated at one or more positions. Carbohydrates, asused herein, encompass unmodified carbohydrates, carbohydratederivatives, substituted carbohydrates, and modified carbohydrates. Asused herein, the phrases “carbohydrate derivatives”, “substitutedcarbohydrate”, and “modified carbohydrates” are synonymous. Modifiedcarbohydrate means any carbohydrate wherein at least one atom has beenadded, removed, or substituted, or combinations thereof. Thus,carbohydrate derivatives or substituted carbohydrates includesubstituted and unsubstituted monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The carbohydrate derivatives orsubstituted carbohydrates optionally can be deoxygenated at anycorresponding C-position, and/or substituted with one or more moietiessuch as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino,amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl,sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl,phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino,hydrazino, carbamyl, phospho, phosphonato, or any other viablefunctional group provided the carbohydrate derivative or substitutedcarbohydrate functions to improve the sweet taste of the sweetenercomposition.

Examples of carbohydrates which may be used in accordance with thisinvention include, but are not limited to, tagatose, trehalose,galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides,various types of maltodextrins, dextran, sucrose, glucose, ribulose,fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose,idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose,isomaltulose, erythrose, deoxyribose, gulose, idose, talose,erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin,glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid,glucono-lactone, abequose, galactosamine, beet oligosaccharides,isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and thelike), xylo-oligosaccharides (xylotriose, xylobiose and the like),xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose,gentiotriose, gentiotetraose and the like), sorbose,nigero-oligosaccharides, palatinose oligosaccharides,fructooligosaccharides (kestose, nystose and the like), maltotetraol,maltotriol, malto-oligosaccharides (maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose and the like), starch,inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose,isomerized liquid sugars such as high fructose corn syrups, couplingsugars, and soybean oligosaccharides. Additionally, the carbohydrates asused herein may be in either the D- or L-configuration.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X obtained according to this invention can be used in combinationwith various physiologically active substances or functionalingredients. Functional ingredients generally are classified intocategories such as carotenoids, dietary fiber, fatty acids, saponins,antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols,plant sterols and stanols (phytosterols and phytostanols); polyols;prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols;amino acids; proteins; vitamins; and minerals. Functional ingredientsalso may be classified based on their health benefits, such ascardiovascular, cholesterol-reducing, and anti-inflammatory.

Highly purified target steviol glycoside(s), particularly, reb D and/orreb X obtained according to this invention may be applied as a highintensity sweetener to produce zero calorie, reduced calorie or diabeticbeverages and food products with improved taste characteristics. It mayalso be used in drinks, foodstuffs, pharmaceuticals, and other productsin which sugar cannot be used. In addition, highly purified targetsteviol glycoside(s), particularly, reb D and/or reb X can be used as asweetener not only for drinks, foodstuffs, and other products dedicatedfor human consumption, but also in animal feed and fodder with improvedcharacteristics.

Examples of products in which highly purified target steviolglycoside(s), particularly, reb D and/or reb X may be used as asweetening compound include, but are not limited to, alcoholic beveragessuch as vodka, wine, beer, liquor, and sake, etc.; natural juices;refreshing drinks; carbonated soft drinks; diet drinks; zero caloriedrinks; reduced calorie drinks and foods; yogurt drinks; instant juices;instant coffee; powdered types of instant beverages; canned products;syrups; fermented soybean paste; soy sauce; vinegar; dressings;mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce;powdered vinegar; types of biscuits; rice biscuit; crackers; bread;chocolates; caramel; candy; chewing gum; jelly; pudding; preservedfruits and vegetables; fresh cream; jam; marmalade; flower paste;powdered milk; ice cream; sorbet; vegetables and fruits packed inbottles; canned and boiled beans; meat and foods boiled in sweetenedsauce; agricultural vegetable food products; seafood; ham; sausage; fishham; fish sausage; fish paste; deep fried fish products; dried seafoodproducts; frozen food products; preserved seaweed; preserved meat;tobacco; medicinal products; and many others. In principle it can haveunlimited applications.

During the manufacturing of products such as foodstuffs, drinks,pharmaceuticals, cosmetics, table top products, and chewing gum, theconventional methods such as mixing, kneading, dissolution, pickling,permeation, percolation, sprinkling, atomizing, infusing and othermethods may be used.

Moreover, the highly purified target steviol glycoside(s), particularly,reb D and/or reb X obtained in this invention may be used in dry orliquid forms. It can be added before or after heat treatment of foodproducts. The amount of the highly purified target steviol glycoside(s),particularly, reb D and/or reb X depends on the purpose of usage. Asdiscussed above, it can be added alone or in combination with othercompounds.

The following examples illustrate preferred embodiments of the inventionfor the preparation of highly purified target steviol glycoside(s),particularly, reb D and/or reb X It will be understood that theinvention is not limited to the materials, proportions, conditions andprocedures set forth in the examples, which are only illustrative.

Example 1 In-Vivo Production of UGT76G1

NcoI and NdeI restriction sides were added to the original nucleicsequence as described in Genbank accession no. AAR06912.1. After codonoptimization the following nucleic sequence (SEQ ID NO. 1) was obtained:

Sequence Listing Free Text

CCATGGCCCATATGGAAAACAAAACCGAAACCACCGTTCGTCGTCGTCGCCGTATTATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCTGCAGCTGGCAAATGTGCTGTATAGCAAAGGTTTTAGCATTACCATTTTTCATACCAATTTTAACAAACCGAAAACCAGCAATTATCCGCATTTTACCTTTCGCTTTATTCTGGATAATGATCCGCAGGATGAACGCATTAGCAATCTGCCGACACATGGTCCGCTGGCAGGTATGCGTATTCCGATTATTAACGAACATGGTGCAGATGAACTGCGTCGTGAACTGGAACTGCTGATGCTGGCAAGCGAAGAAGATGAAGAAGTTAGCTGTCTGATTACCGATGCACTGTGGTATTTTGCACAGAGCGTTGCAGATAGCCTGAATCTGCGTCGTCTGGTTCTGATGACCAGCAGCCTGTTTAACTTTCATGCACATGTTAGCCTGCCGCAGTTTGATGAACTGGGTTATCTGGATCCGGATGATAAAACCCGTCTGGAAGAACAGGCAAGCGGTTTTCCGATGCTGAAAGTGAAAGATATCAAAAGCGCCTATAGCAATTGGCAGATTCTGAAAGAAATTCTGGGCAAAATGATTAAACAGACCAAAGCAAGCAGCGGTGTTATTTGGAATAGCTTTAAAGAACTGGAAGAAAGCGAACTGGAAACCGTGATTCGTGAAATTCCGGCACCGAGCTTTCTGATTCCGCTGCCGAAACATCTGACCGCAAGCAGCAGCAGCCTGCTGGATCATGATCGTACCGTTTTTCAGTGGCTGGATCAGCAGCCTCCGAGCAGCGTTCTGTATGTTAGCTTTGGTAGCACCAGCGAAGTTGATGAAAAAGATTTTCTGGAAATTGCCCGTGGTCTGGTTGATAGCAAACAGAGCTTTCTGTGGGTTGTTCGTCCGGGTTTTGTTAAAGGTAGCACCTGGGTTGAACCGCTGCCGGATGGTTTTCTGGGTGAACGTGGTCGTATTGTTAAATGGGTTCCGCAGCAAGAAGTTCTGGCACACGGCGCAATTGGTGCATTTTGGACCCATAGCGGTTGGAATAGCACCCTGGAAAGCGTTTGTGAAGGTGTTCCGATGATTTTTAGCGATTTTGGTCTGGATCAGCCGCTGAATGCACGTTATATGAGTGATGTTCTGAAAGTGGGTGTGTATCTGGAAAATGGTTGGGAACGTGGTGAAATTGCAAATGCAATTCGTCGTGTTATGGTGGATGAAGAAGGTGAATATATTCGTCAGAATGCCCGTGTTCTGAAACAGAAAGCAGATGTTAGCCTGATGAAAGGTGGTAGCAGCTATGAAAGCCTGGAAAGTCTGGTTAGCTATATTAGCAGCCTGTAATAACTCGAG

After synthesis of the gene and subcloning into pET30A+ vector usingNdeI and XhoI cloning sites, the UGT76G1_pET30a+ plasmid was introducedin E. coli B121(DE3) and E. coli EC100 by electroporation. The obtainedcells were grown in petri-dishes in the presence of Kanamycin andsuitable colonies were selected and allowed to grow in liquid LB medium(erlenmeyer flasks). Glycerol was added to the suspension ascryoprotectant and 400 μL aliquots were stored at −20° C. and at −80° C.

The storage aliquots of E. coli BL21(DE3) containing the pET30A+_UGT76G1plasmid were thawed and added to 30 mL of LBGKP medium (20 g/L LuriaBroth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM Phosphate buffer pH7.00; 2.5 g/L glucose and 50 mg/L of Kanamycin). This culture wasallowed to shake at 135 rpm at 30° C. for 8 h.

The production medium contained 60 g/L of overnight express instant TBmedium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycin. Themedium was allowed to stir at 20° C. while taking samples to measure theOD and pH. The cultures gave significant growth and a good OD wasobtained. After 40 h, the cells were harvested by centrifugation andfrozen to yield 12.7 g of cell wet weight.

Lysis was performed by addition of Bugbuster Master mix (Novagen) andthe lysate was recovered by centrifugation and kept frozen. Activitytests were performed with thawed lysate.

Example 2 In-Vitro Production of UGT76G1

The S30 T7 High Yield Protein expression system kit from Promega wasused. 4 μs of UGT76G1pET30a+ plasmid from E. coli EC100 was mixed with80 μL of S30 premix plus and 72 μL of S30 T7 extract was added.Nuclease-free water was added in order to obtain a total volume of 200μL and the resulting solution was incubated for 2 h at 30° C. 180 μL wasused in the catalytic test reaction.

Example 3 In-Vitro Production of UGT91D2

NcoI and NdeI restriction sides were added to the original nucleicsequence as described in Genbank accession no. ACE87855.1. After codonoptimization the following nucleic sequence (SEQ ID NO. 2) was obtained:

Sequence Listing Free Text

CCATGGCACATATGGCAACCAGCGATAGCATTGTTGATGATCGTAAACAGCTGCATGTTGCAACCTTTCCGTGGCTGGCATTTGGTCATATTCTGCCGTATCTGCAGCTGAGCAAACTGATTGCAGAAAAAGGTCATAAAGTGAGCTTTCTGAGCACCACCCGTAATATTCAGCGTCTGAGCAGCCATATTAGTCCGCTGATTAATGTTGTTCAGCTGACCCTGCCTCGTGTTCAAGAACTGCCGGAAGATGCCGAAGCAACCACCGATGTTCATCCGGAAGATATTCCGTATCTGAAAAAAGCAAGTGATGGTCTGCAGCCGGAAGTTACCCGTTTTCTGGAACAGCATAGTCCGGATTGGATCATCTATGATTATACCCATTATTGGCTGCCGAGCATTGCAGCAAGCCTGGGTATTAGCCGTGCACATTTTAGCGTTACCACCCCGTGGGCAATTGCATATATGGGTCCGAGCGCAGATGCAATGATTAATGGTAGTGATGGTCGTACCACCGTTGAAGATCTGACCACCCCTCCGAAATGGTTTCCGTTTCCGACCAAAGTTTGTTGGCGTAAACATGATCTGGCACGTCTGGTTCCGTATAAAGCACCGGGTATTAGTGATGGTTATCGTATGGGTCTGGTTCTGAAAGGTAGCGATTGTCTGCTGAGCAAATGCTATCATGAATTTGGCACCCAGTGGCTGCCGCTGCTGGAAACCCTGCATCAGGTTCCGGTTGTTCCGGTGGGTCTGCTGCCTCCGGAAGTTCCGGGTGATGAAAAAGATGAAACCTGGGTTAGCATCAAAAAATGGCTGGATGGTAAACAGAAAGGTAGCGTGGTTTATGTTGCACTGGGTAGCGAAGTTCTGGTTAGCCAGACCGAAGTTGTTGAACTGGCACTGGGTCTGGAACTGAGCGGTCTGCCGTTTGTTTGGGCATATCGTAAACCGAAAGGTCCGGCAAAAAGCGATAGCGTTGAACTGCCGGATGGTTTTGTTGAACGTACCCGTGATCGTGGTCTGGTTTGGACCAGCTGGGCACCTCAGCTGCGTATTCTGAGCCATGAAAGCGTTTGTGGTTTTCTGACCCATTGTGGTAGCGGTAGCATTGTGGAAGGTCTGATGTTTGGTCATCCGCTGATTATGCTGCCGATTTTTGGTGATCAGCCGCTGAATGCACGTCTGCTGGAAGATAAACAGGTTGGTATTGAAATTCCGCGTAATGAAGAAGATGGTTGCCTGACCAAAGAAAGCGTTGCACGTAGCCTGCGTAGCGTTGTTGTTGAAAAAGAAGGCGAAATCTATAAAGCCAATGCACGTGAACTGAGCAAAATCTATAATGATACCAAAGTGGAAAAAGAATATGTGAGCCAGTTCGTGGATTATCTGGAAAAAAACACCCGTGCAGTTGCCATTGATCACGAAAGCTAATGACTCGAG

After synthesis of the gene and subcloning into pET30A+ vector usingNcoI and XhoI cloning sites, the UGT91D2_pET30a+ plasmid was introducedinto E. coli EC100 by electroporation. The obtained cells were grown inthe presence of Kanamycin and suitable colonies were selected andallowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol wasadded to the suspension as cryoprotectant and 400 μL aliquots werestored at −20° C. and at −80° C.

The S30 T7 High Yield Protein expression system kit from Promega wasused for the in-vitro synthesis of the protein.

4 μg of UGT91D2_pET30a+ plasmid was mixed with 80 μL of S30 premix plusand 72 μL of S30 T7 extract was added. Nuclease-free water was added inorder to obtain a total volume of 200 μL and the resulting solution wasincubated for 2 h at 30° C. 5 μL was used for SDS-page analysis whilethe remaining 45 μL, was used in the catalytic test reaction.

Example 4 Catalytic Reaction with In-Vivo Produced UGT76G1

The total volume of the reaction was 5.0 mL with the followingcomposition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl₂, 2.5 mMUDP-glucose, 0.5 mM Stevioside and 500 μL of UGT76G1 thawed lysate. Thereactions were run at 30° C. on an orbitary shaker at 135 rpm. For eachsample, 460 μL of the reaction mixture was quenched with 40 μL of 2NH₂SO₄ and 420 μL of methanol/water (6/4). The samples were immediatelycentrifuged and kept at 10° C. before analysis by HPLC (CAD). HPLCindicated almost complete conversion of stevioside to rebaudioside A.

Example 5 Catalytic Reaction with In-Vitro Produced UGT91D2

The total volume of the reaction was 0.5 mL with the followingcomposition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl₂, 3.8 mMUDP-glucose, 0.1 mM Rebaudioside A and 180 μL of in-vitro producedUGT91D2. The reactions were run at 30° C. on an orbitary shaker at 135rpm. For each sample, 450 μL of reaction mixture was quenched with 45 μLof 2N H₂SO₄ and 405 μL of 60% MeOH. After centrifugation, thesupernatant was analyzed by HPLC (CAD). HPLC indicated a 4.7% conversionof rebaudioside A to rebaudioside D after 120 h.

Example 6 Catalytic Reaction with In-Vitro Produced UGT76G1

The total volume of the reaction was 2 mL with the followingcomposition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl₂, 3.8 mMUDP-glucose, 0.5 mM Rebaudioside D and 180 μL of in-vitro producedUGT76G1. The reactions were run at 30° C. on an orbitary shaker at 135rpm. For each sample, 400 μL, of reaction mixture was quenched with 40μL of 2N H₂SO₄ and 360 μL of 60% MeOH. After centrifugation, thesupernatant was analyzed by HPLC (CAD). HPLC indicated 80% conversion ofrebaudioside D to rebaudioside X after 120 h.

For examples 7 to 12, the following abbreviations were used:LBGKP medium: 20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50mMPhosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of Kanamycin orAmpicillinLB medium: (20 g/L Luria Broth Lennox)

Example 7 Preparation and Activity of UGT76G1 Prepared by pET30a+Plasmidand BL21 (DE3) Expression Strain

The pET30a+_UGT76G1 plasmid was transformed into BL21(DE3) expressionstrain (Lucigen E. Cloni® EXPRESS Electrocompetent Cells). The obtainedcells were grown on LB Agar medium in petri-dishes in the presence ofKanamycin. Suitable colonies were selected and allowed to grow in liquidLBGKP medium containing Kanamycin. Glycerol was added and 400 μLaliquots were stored at −20° C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. Thisculture was allowed to shake at 30° C. for 8 h. and subsequently used toinoculate 400 mL of production medium containing 60 g/L of “Overnightexpress instant TB medium” (Novagen, reference 71491-5), 10 g/L ofglycerol and 50 mg/L of Kanamycin. The medium was allowed to stir at 20°C. while taking samples to measure the OD (600 nm) and pH. After 40 h,the cells were harvested by centrifugation and frozen. The obtained cellwet weight was 10.58 g.

3.24 g of obtained pellet was lysed by addition of 8.1 mL of “BugbusterMaster mix” (Novagen, reference 71456) and 3.5 mL of water. The lysatewas recovered by centrifugation and kept frozen.

Example 8 Preparation and Activity of UGT76G1 Prepared by pET30a+Plasmid and Tuner (DE3) Expression Strain

The pET30a-LUGT76G1 plasmid was transformed into Tuner (DE3) expressionstrain (Novagen Tuner™ (DE3) Competent cells) by heat shock treatment.The obtained cells were grown on LB Agar medium in petri-dishes in thepresence of Kanamycin. Suitable colonies were selected and allowed togrow in liquid LBGKP medium containing Kanamycin). Glycerol was addedand 400 μL aliquots were stored at −20° C. and at −80° C.

A storage aliquot was thawed and added to 100 mL of LB medium containing50 mg/L of Kanamycin. This culture allowed to shake at 30° C. for 15 h.4.4 mL of this culture was used to inoculate 200 mL of production mediumcontaining LB. This medium was allowed to stir at 37° C. until an OD(600 nm) of 0.9 was obtained, after which 400 μL of a 100 mM IPTGsolution was added and the medium was allowed to stir at 30° C. for 4 h.The cells were harvested by centrifugation and frozen. The obtained cellwet weight was 1.38 g.

The obtained pellet was lysed by addition of 4.9 mL of “Bugbuster Mastermix” (Novagen, reference 71456) and 2.1 mL of water. The lysate wasrecovered by centrifugation and kept frozen.

Example 9 Preparation and Activity of UGT76G1 Prepared by pMAL Plasmidand BL21 Expression Strain

After subcloning the synthetic UGT76G1 gene into the pMAL plasmid usingNde1 and Sal1 cloning sites, the pMAL_UGT76G1 plasmid was transformedinto BL21 expression strain (New England Biolabs BL21 Competent E. coli)by heat shock treatment. The obtained cells were grown on LB Agar mediumin petri-dishes in the presence of Ampicillin. Suitable colonies wereselected and allowed to grow in liquid LBGKP medium containingAmpicillin). Glycerol was added and 400 μL aliquots were stored at −20°C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. Thisculture was allowed to shake at 30° C. for 8 h. and subsequently used toinoculate 400 mL of production medium containing 60 g/L of “Overnightexpress instant TB medium” (Novagen, reference 71491-5), 10 g/L ofglycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at20° C. while taking samples to measure the OD and pH.

After 40 h, the cells were harvested by centrifugation and frozen. Theobtained cell wet weight was 5.86 g.

2.74 g of obtained pellet was lysed by addition of 9.6 mL of “BugbusterMaster Mix” (Novagen, reference 71456) and 4.1 mL of water. The lysatewas recovered by centrifugation and kept frozen.

Example 10 Preparation and Activity of UGT76G1 Prepared by pMAL Plasmidand ArcticExpress Expression Strain

The pMAL_UGT76G1 plasmid was transformed into ArcticExpress expressionstrain (Agilent ArcticExpress competent cells) by heat shock treatment.The obtained cells were grown on LB Agar medium in petri-dishes in thepresence of Ampicillin and Geneticin. Suitable colonies were selectedand allowed to grow in liquid LBGKP medium containing of Ampicillin andGeneticin. Glycerol was added and 400 μL aliquots were stored at −20° C.and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium(containing Ampicillin and Geneticin). This culture was allowed to shakeat 30° C. for 8 h. and subsequently used to inoculate 400 mL ofproduction medium containing 60 g/L of “Overnight express instant TBmedium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L ofAmpicillin. The medium was allowed to stir at 12° C. while takingsamples to measure the OD (600 nm) and pH. After 68 h, the cells wereharvested by centrifugation and frozen. The obtained cell wet weight was8.96 g.

2.47 g of the obtained pellet was lysed by addition of 8.73 mL of“Bugbuster Master Mix” (Novagen, reference 71456) and 3.79 mL of water.The lysate was recovered by centrifugation and kept frozen.

Example 11 Preparation and Activity of UGT76G1 Prepared by pCOLDIIIPlasmid and ArcticExpress Expression Strain

After subcloning the synthetic UGT76G1 gene into the pCOLDIII plasmidusing Nde1 and Xho1 cloning sites, the pCOLDIII_UGT76G1 plasmid wastransformed into ArcticExpress expression strain (Agilent ArcticExpresscompetent cells) by heat shock treatment. The obtained cells were grownon LB Agar medium in petri-dishes in the presence of Ampicillin andGeneticin. Suitable colonies were selected and allowed to grow in liquidLBGKP medium containing Ampicillin and Geneticin. Glycerol was added and400 μL aliquots were stored at −20° C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium(containing Ampicillin and Geneticin). This culture was allowed to shakeat 30° C. for 8 h. and subsequently used to inoculate 400 mL ofproduction medium containing 60 g/L of “Overnight express instant TBmedium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L ofKanamycin. The medium was allowed to stir at 12° C. while taking samplesto measure the OD (600 nm) and pH. After 63 h, the cells were harvestedby centrifugation and frozen. The obtained cell wet weight was 6.54 g.

2.81 g of the obtained pellet was lysed by addition of 9.8 mL of“Bugbuster Master Mix” (Novagen, reference 71456) and 4.2 mL of water.The lysate was recovered by centrifugation and kept frozen.

Example 12 Preparation and Activity of UGT76G1 Prepared by pCOLDIIIPlasmid and Origami2 (DE3) Expression Strain

The pCOLDIII_UGT76G1 plasmid was transformed into Origami2 (DE3)expression strain (Novagen Origami™2 (DE3) Competent Cells) by heatshock treatment. The obtained cells were grown on LB Agar medium inpetri-dishes in the presence of Ampicillin. Suitable colonies wereselected and allowed to grow in liquid LBGKP medium containingAmpicillin. Glycerol was added and 400 μL aliquots were stored at −20°C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium(containing Ampicillin). This culture was allowed to shake at 30° C. for8 h. and subsequently used to inoculate 400 mL of production mediumcontaining 60 g/L of “Overnight express instant TB medium” (Novagen,reference 71491-5), 10 g/L of glycerol and 50 mg/L of Kanamycin. Themedium was allowed to stir at 12° C. while taking samples to measure theOD (600 nm) and pH. After 68 h, the cells were harvested bycentrifugation and frozen. The obtained cell wet weight was 2.53 g.

1.71 g of the obtained pellet was lysed by addition of 6.0 mL of“Bugbuster Master mix” (Novagen, reference 71456) and 1.9 mL of water.The lysate was recovered by centrifugation and kept frozen.

Example 13 Determination of Activity

Activity tests were performed on a 5 mL scale with 500 μL of thawedlysate for the transformation of Stevioside to Rebaudioside A andRebaudioside D to Rebaudioside X using 0.5 mM of substrate, 2.5 mM ofUDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2.Samples were taken and analyzed by HPLC. The results for the differentpreparations of UGT76G1 are summarized in the following table.

Transformation activity* Rebaud- Stevioside ioside D Expression toRebaud- to Rebaud- Example Plasmid strain ioside A ioside X 7 pET30a+BL21 (DE3) 29 U mL⁻¹ 0.31 U mL⁻¹ 8 pET30a+ Tuner (DE3) 33 U mL⁻¹ 0.40 UmL⁻¹ 9 pMAL BL21 20 U mL⁻¹ 0.15 U mL⁻¹ 10 pMAL ArcticExpress 15 U mL⁻¹0.25 U mL⁻¹ 11 pCOLDIII ArcticExpress 15 U mL⁻¹ 0.11 U mL⁻¹ 12 pCOLDIIIOrigami2 (DE3) 37 U mL⁻¹ 0.20 U mL⁻¹ *Note The activities for thetransformation of Stevioside and Rebaudioside X are mentioned per mL oflysate. 1 U will transform 1 μmol of substrate in 1 hour at 30° C. andpH 7.2

Example 14 50 mL Scale Reaction for the Transformation of Rebaudioside Dto Rebaudioside X

5 mL of the lysate of Example 12 was used to transform Rebaudioside D toRebaudioside X on a 50 mL scale. The reaction medium consisted of 50 mMSodium Phosphate buffer pH 7.2, 3 mM of MgCl₂, 2.5 mM of UDP-Glucose and0.5 mM of Rebaudioside D. After allowing the reaction to be shaken at30° C. for 90 h. 50 mL of ethanol was added and the resulting mixturewas allowed to stir at −20° C. for 1 h. After centrifugation at 5000 gfor 10 min. the supernatant was purified via ultrafiltration (VivaflowMWCO 30000). 78 mL of permeate was obtained and the 9 mL of retentatewas diluted with 9 mL of ethanol and resubjected to Ultrafiltration(Vivaflow MWCO 30000). Another 14 mL of filtrate was obtained, which wascombined with the first permeate. The combined permeates wereconcentrated under reduced pressure at 30° C. until 32 mL of a clearsolution was obtained.

The HPLC trace of the product mixture is shown in FIG. 5. HPLC wascarried out on an Agilent 1200 series equipped with a binary pump, autosampler, and thermostat column compartment. The method was isocratic,with a mobile phase composed of 70% water (0.1% formic acid): 30%acetonitrile. The flow rate was 0.1 μL/min. The column used wasPhenomenex Prodigy 5μODS (3) 100 A; 250x2 mm. The column temperature wasmaintained at 40° C. The injection volume was 20-40

Example 15 Preparation of UGT91D2 Using pMAL Plasmid and BL21 ExpressionStrain

After subcloning the synthetic UGT91D2 gene into the pMAL plasmid usingNde1 and Sal1 cloning sites, the pMAL_UGT91D2 plasmid was transformedinto BL21 expression strain (New England Biolabs BL21 Competent E. coli)by heat shock treatment. The obtained cells were grown on LB Agar mediumin petri-dishes in the presence of Ampicillin. Suitable colonies wereselected and allowed to grow in liquid LBGKP medium containingAmpicillin). Glycerol was added and 400 μL, aliquots were stored at −20°C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. Thisculture was allowed to shake at 30° C. for 8 h. and subsequently used toinoculate 400 mL of production medium containing 60 g/L of “Overnightexpress instant TB medium” (Novagen, reference 71491-5), 10 g/L ofglycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at20° C. while taking samples to measure the OD and pH. After 40 h, thecells were harvested by centrifugation and frozen. The obtained cell wetweight is 12.32 g.

2.18 g of obtained pellet was lysed by addition of 7.7 mL of “BugbusterMaster Mix” (Novagen, reference 71456) and 3.2 mL of water. The lysatewas recovered by centrifugation and used directly for activity testing.

Example 16 Preparation of UGT91D2 Using pMAL Plasmid and ArcticExpressExpression Strain

The pMAL_UGT91D2 plasmid was transformed into ArcticExpress expressionstrain (Agilent ArcticExpress competent cells) by heat shock treatment.The obtained cells were grown on LB Agar medium in petri-dishes in thepresence of Ampicillin and Geneticin. Suitable colonies were selectedand allowed to grow in liquid LBGKP medium containing Ampicillin andGeneticin. Glycerol was added and 400 μL aliquots were stored at −20° C.and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium(containing Ampicillin and Geneticin). This culture was allowed to shakeat 30° C. for 8 h. and subsequently used to inoculate 400 mL ofproduction medium containing 60 g/L of “Overnight express instant TBmedium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L ofAmpicillin. The medium was allowed to stir at 20° C. for 16 h. followedby another 50 h. at 12° C. while taking samples to measure the OD (600nm) and pH. The cells were harvested by centrifugation and frozen. Theobtained cell wet weight is 15.77 g.

2.57 g of the obtained pellet was lysed by addition of 9.0 mL of“Bugbuster Master Mix” (Novagen, reference 71456) and 3.8 mL of water.The lysate was recovered by centrifugation and used directly foractivity testing.

Example 17 Preparation of UGT91D2 Using pET30a+ Plasmid and Tuner (DE3)Expression Strain

The pET30a+_UGT91D2 plasmid was transformed into Tuner (DE3) expressionstrain (Novagen Tuner™ (DE3) Competent cells) by heat shock treatment.The obtained cells were grown on LB Agar medium in petri-dishes in thepresence of Kanamycin. Suitable colonies were selected and allowed togrow in liquid LBGKP medium (containing Kanamycin). Glycerol was addedand 400 μL aliquots were stored at −20° C. and at −80° C.

A storage aliquot was thawed and added to 100 mL of LB medium containing50 mg/L of Kanamycin. This culture allowed to shake at 30° C. for 15 h.6.2 mL of this culture was used to inoculate 500 mL of production mediumcontaining LB. This medium was allowed to stir at 37° C. until an OD(600 nm) of 0.9 was obtained after which 500 μL of a 100 mM IPTGsolution was added (IPTG concentration in medium is 100 μM) and themedium was allowed to stir at 30° C. for 4 h, the cells were harvestedby centrifugation and frozen. The obtained cell wet weight is 4.02 g.

1.92 g of the obtained pellet was lysed by addition of 6.8 mL of“Bugbuster Master mix” (Novagen, reference 71456) and 2.8 mL of water.The lysate was recovered by centrifugation and tested directly foractivity.

Example 18 Preparation of UGT91D2 Using pET30a+ Plasmid andArcticExpress Expression Strain

The pET30a+_UGT91D2 plasmid was transformed into ArcticExpress (DE3)expression strain (Agilent ArcticExpress competent cells) by heat shocktreatment. The obtained cells were grown on LB Agar medium inpetri-dishes in the presence of Kanamycin and Geneticin. Suitablecolonies were selected and allowed to grow in liquid LBGKP mediumcontaining of Kanamycin and Geneticin. Glycerol was added and 400 μLaliquots were stored at −20° C. and at −80° C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium(containing Kanamycin and Geneticin). This culture was allowed to shakeat 30° C. for 8 h. and subsequently used to inoculate 400 mL ofproduction medium containing 60 g/L of “Overnight express instant TBmedium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L ofAmpicillin. The medium was allowed to stir at 20° C. for 16 h. followedby another 50 h. at 12° C. while taking samples to measure the OD (600nm) and pH. After 60 h, the cells were harvested by centrifugation andfrozen. The obtained cell wet weight is 16.07 g.

3.24 g of the obtained pellet was lysed by addition of 11.4 mL of“Bugbuster Master Mix” (Novagen, reference 71456) and 4.8 mL of water.The lysate was recovered by centrifugation and used directly foractivity testing.

Example 19 Determination of Activity of In-Vivo Preparations of UGT91D2

Activity tests were performed at 5 mL scale with 1000 μL of lysate forthe transformation of Rubusoside to Stevioside using 0.5 mM ofsubstrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM SodiumPhosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. Theresults for the different preparations of UGT91D2 are summarized in thefollowing table.

Transformation activity* Example Plasmid Expression strain Rubusoside toStevioside 15 pMAL BL21  9 mU mL⁻¹ 16 pMAL ArcticExpress 60 mU mL⁻¹ 17pET30a+ Tuner (DE3) 28 mU mL⁻¹ 18 pET30a+ ArcticExpress (DE3) 21 mU mL⁻¹*Note: The activities are mentioned per mL of lysate. 1 U will transform1 μmol of substrate in 1 hour at 30° C. and pH 7.2

Example 20 Isolation of Rebaudioside X

The amount of the product mixture of Example 14 was not large enough toseparate via preparative HPLC methods. Accordingly, analytical HPLC witha series of injections was used to separate the components of themixture. Separation was conducted according to the method describedabove in Example 14 to provide two fractions corresponding to the twomain peaks in the HPLC trace of FIG. 5: Fraction A (retention time24.165 minutes) and Fraction B (retention time 31.325 minutes).

The retention time of Fraction A was consistent with reb D, indicatingunreacted starting material from the biotransformation reaction.

The retention time of purified Fraction B (FIG. 6) was consistent withreb X, indicating successful biotransformation from reb D. The identityof the material collected in Fraction B as reb X was confirmed byco-injection of purified Fraction B with a reb X standard (availablefrom Pure Circle, HPLC trace of reb X standard shown in FIG. 7). BothFraction B and the reb X standard were found to elute at the sameretention time (FIG. 8), indicating Fraction B was reb X.

The identity of Fraction B as reb X was also separately confirmed by NMRand HRMS. For sampling, Fraction B was concentrated under rotovapor,freeze dried and dried for 40 h at 40° C.

The NMR sample was dissolved in deuterated pyridine (C₅D₅N) and spectrawere acquired on a Varian Unity Plus 600 MHz instrument using standardpulse sequences. The NMR spectra of Fraction B was compared to the NMRspectra of reb X. An overlay of the two spectra (FIG. 9) showedconsistency of peaks of Fraction B with reb X. A table of the NMRassignments for reb X is shown below:

¹H and ¹³C NMR spectral data for Rebaudioside X in C₅D₅N^(a-c). Position¹³C NMR ¹H NMR 1 40.3 0.75 t (13.2) 1.76 m 2 19.6 1.35 m 2.24 m 6′ 61.84.20 m 4.31 m 1″ 96.2 5.46 d (7.1) 2″ 81.4 4.13 m 3″ 87.9 4.98 t (8.5)4″ 70.4 4.07 t (9.6) 5″ 77.7 3.94 m 6″ 62.6 4.19 m 4.32 m 1′″ 104.8 5.48d (7.7) 2′″ 75.8 4.15 m 3′″ 78.6 4.13 m 4′″ 73.2 3.98 m 5′″ 77.6 3.74ddd (2.8, 6.4, 9.9) 6′″ 64.0 4.27 m 4.51m 1″″ 103.9 5.45 d (7.5) 2″″75.6 3.98 m 3″″ 77.8 4.50 t (7.8) 4″″ 71.3 4.14 m 5″″ 78.0 3.99 m 6″″62.1 4.20 m 4.32 m 1″″′ 104.2 5.81 d (7.2) 2″″′ 75.5 4.20 m 3″″′ 78.44.20 m 4″″′ 73.6 4.10 m 5″″′ 77.8 3.90 ddd (2.8, 6.4, 9.9) 6″″′ 64.04.32 m 4.64 d (10.3) 1″″″ 104.1 5.31 d (8.0) 2″″″ 75.5 3.95 m 3″″″ 78.04.37 t (9.1) 4″″″ 71.1 4.10 m 5″″″ 78.1 3.85 ddd (1.7, 6.1, 9.9) 6″″″62.1 4.10 m 4.32 ^(a)assignments made on the basis of COSY, HMQC andHMBC correlations; ^(b)Chemical shift values are in δ (ppm);^(c)Coupling constants are in Hz.

HRMS (FIG. 10) was generated with a Waters Premier QuadropoleTime-of-Flight (Q-TOF) mass spectrometer equipped with an electrosprayionization source operated in the positive-ion mode. The sample wasdissolved in methanol and eluted in 2:2:1 methanol: acetonitrile: waterand introduced via infusion using the onboard syringe pump. The presenceof reb X was confirmed by a [M+Na]⁺ adduct at m/z 1313.5265, whichcorresponds to a molecular formula of C₅₆H₉₀O₃₃:

1-26. (canceled)
 27. A method for making Rebaudioside X comprisingconverting Rebaudioside D to Rebaudioside X using aUDP-glucosyltransferase.
 28. Rebaudioside X made by the method of claim27 and having a purity of greater than about 80% by weight. 29.Rebaudioside X made by the method of claim 27 and having a purity ofgreater than about 90% by weight.
 30. Rebaudioside X made by the methodof claim 27 and having a purity of greater than about 95% by weight. 31.A consumable product comprising the Rebaudioside X made by the method ofclaim
 27. 32. The method of claim 27, wherein theUDP-glucosyltransferase comprises any UDP-glucosyltransferase capable ofadding at least one glucose unit to Rebaudioside D to form RebaudiosideX.
 33. The method of claim 32, wherein the UDP-glucosyltransferase iscapable of adding a glucose unit to a C-19 position of Rebaudioside D.